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Clonality analysis is used to test malignant transformation and tumour progression. X-chromosome linked clonality assays have been employed for this purpose, but are subject to certain technical limitations. This paper reviews the issues involved and the controls that are necessary to ensure valid interpretation of such analyses.
Designing a molecular analysis of clonality in tumours
Salvador J. Diaz-Cano
Department of Histopathology and Morbid Anatomy, St Bartholomew's and the Royal London School of Medicine and Dentistry, Whitechapel, London
E1 1BB, UK
Clonality analysis is used to test malignant transformation and tumour progression. X-chromosome
linked clonality assays have been employed for this purpose, but are subject to certain technical
limitations. This paper reviews the issues involved and the controls that are necessary to ensure
valid interpretation of such analyses.
Keywords: clonality; X-chromosome; lyonization; neoplasia
Clonality is an essential attribute of neoplasms and its
analysis has been used to test malignant transforma-
tion and tumour progression [1,2]. Concordant pat-
terns of genetic markers (X-linked or not) in different
tumours suggest that a common progenitor contribu-
ted to those lesions and favour, therefore, a multifocal
rather than a multicentric origin. These shared genetic
alterations also suggest a common cellular origin for
biphasic neoplasms [3]. Saxena et al. recently reported
a monoclonal pattern in smooth muscle cells and blood
vessels of sporadic angiomyolipoma, while the adipose
tissue revealed a polyclonal pattern [4]. Based on these
®ndings the authors concluded that the polyclonal
adipose tissue is probably metaplastic or reactive.
This represents a good example of the application of
clonality in tumour biology.
However, some biological and technical issues arise
from this article. X-linked clonality assays are based on
DNA polymorphism and random X-chromosome
inactivation (XCI) in females. Those features enable
us to distinguish the maternally from the paternally
inherited X-chromosomes [1,5,6]. The mechanisms
leading to XCI have not been fully characterized, but
DNA methylation might maintain the inactive state,
once it is established during early embryogenesis.
These methylation patterns are then transmitted by
clonal inheritance through the strong preference of
mammalian DNA (cytosine-5)-methyltransferase for
hemimethylated DNA, involving the promoter regions
of alleles on the inactive X-chromosome only [7]. Since
XCI analysis is based on differential DNA methylation
of one allele from X-chromosome genes (e.g. human
androgen receptor gene), suboptimal enzymatic diges-
tion and abnormal methylation can result in changes
of clonality patterns.
According to Lyon's hypothesis, all but one X-
chromosomes in a cell are randomly inactivated during
early embryogenesis, when the primordial cell pool
may comprise as few as 16±30 cells [8]. Given that
small number of embryo-destined cells, it reasonable to
expect unequal numbers of paternally- and maternally-
inherited inactive X-chromosomes, although the X-
chromosome is randomly inactivated in each cell. The
average Lyonization ratio is close to 50 : 50 in large cell
populations, although individual variation has been
found [8]. Skewing towards one allele to an extent that
meets the criteria for clonal derivation is consistent
with early XCI during embryogenesis (Figure 1).
This ®nding leads us to consider the selection of
appropriate controls to assess the Lyonization ratio in
each female. This ratio can also vary from tissue to
tissue in the same individual, due to unequal splitting
of the cells derived from the primordial cell pool, or to
different methylation patterns in different tissues [6,9].
Controls for unequal Lyonization should thus ideally
be the most closely related tissue thought not to be
involved in the disease process. An essential require-
Figure 1. Methylation pattern of androgen receptor alleles in
control samples. Only polymorphic and polyclonal controls (two
allele bands in both undigested and digested samples) are
considered informative for clonality assays (lanes 1 and 2). The
remaining possibilities (lanes 3±8) should be excluded from
clonality analyses, due to either monoclonal origin of controls
(lanes 3±6) or absence of locus polymorphism (lanes 7and 8).
U=undigested sample: D=digested sample
Journal of Pathology
J Pathol 2000; 191: 343±344.
Copyright #2000 John Wiley & Sons, Ltd.
ment for clonality analysis is the identi®cation of a
polymorphic locus in the normal control (Figure 1). In
every case, the tumour sample must be compared with
matched controls from the same patient to test the
heterozygosity for the marker. Additionally, the indi-
vidual variability and tissue-related Lyonization ratio
require samples of close embryological origin. This
feature must be maintained in the digested sample in
those tests based on XCI (Figure 1).
Positive allelic imbalances are determined case-by-
case, using the skewed data normalized by the allele
ratio in matched controls [1,6]. Allelic imbalance
analysis is based on the allele ratio and requires
densitometric analysis of both allele bands. Therefore,
the allele ratio in the target DNA must be maintained
in the ampli®cation product, which has to avoid the
PCR plateau phase. At this level, any PCR ampli®ca-
tion bias should be considered, especially DNA
degradation of the larger allele in formalin-®xed,
paraf®n-embedded tissues and defective ampli®cation
of repetitive CG-rich sequences [10±12].
Early XCI occurs randomly and results in a chess-
board pattern of cells descended from a common
progenitor, which may grow together like a clone
(patch size mosaicism). This pattern represents an
example of tissue heterogeneity that can also be present
in tumours. Sample size is a limiting factor; the lower
the cell number, the higher the probability of mono-
clonal patterns based on patch size mosaicism. This
concept becomes particularly important in mixed
tumours, where multiple microdissected samples from
different tumour areas (i100 cells) and from controls
are required to address the question.
Monoclonal patterns support a neoplastic rather
than a reactive or hyperplastic process, but are not
diagnostic of it. Host cell contamination of tumour
samples could give false heterozygous results that
would require careful microdissection and microscopic
control of the sample collection. However, the pitfalls
mentioned above should be always excluded.
Some of these considerations do not appear to have
been addressed in the paper of Saxena et al. [4],
especially those concerning tests for digestion comple-
tion with restriction endonuclease; controls regarding
both tumour heterogeneity and their methylation
patterns; PCR bias in the ampli®cation of both alleles;
tumour heterogeneity and patch size mosaicism; and
the meaning of monoclonal and polyclonal patterns.
1. Diaz-Cano SJ, Blanes A, Wolfe HJ. PCR-based techniques for
clonality analysis of neoplastic progression. Bases for its
appropriate application in paraf®n-embedded tissues. Diagn
Mol Pathol (in press).
2. Diaz-Cano SJ. Clonality studies in the analysis of adrenal
medullary proliferations: application principles and limitations.
Endocr Pathol 1998; 9: 301±316.
3. Zhuang Z, Lininger RA, Man YG, Albuquerque A, Merino
MJ, Tavassoli FA. Identical clonality of both components
of mammary carcinosarcoma with differential loss of hetero-
zygosity. Mod Pathol 1997; 10: 354±362.
4. Saxena A, Alport EC, Custead S, Skinnider LF. Molecular
analysis of clonality of sporadic angiomyolipoma. J Pathol 1999;
189: 79±84.
5. Sleddens HF, Oostra BA, Brinkmann AO, Trapman J. Tri-
nucleotide repeat polymorphism in the androgen receptor gene
(AR). Nucleic Acids Res 1992; 20: 1427.
6. Mutter GL, Boynton KA. X chromosome inactivation in the
normal female genital tract: implications for identi®cation of
neoplasia. Cancer Res 1995; 55: 5080±5084.
7. Lyon MF. Some milestones in the history of X-chromosome
inactivation. Annu Rev Genet 1992; 26: 16±28.
8. Fialkow PJ. Primordial cell pool size and lineage relationships of
®ve human cell types. Ann Hum Genet 1973; 37: 39±48.
9. Kappler JW. The 5-methylcytosine content of DNA: tissue
speci®city. J Cell Physiol 1971; 78: 33±36.
10. Mutter GL, Boynton KA. PCR bias in ampli®cation of
androgen receptor alleles, a trinucleotide repeat marker used in
clonality studies. Nucleic Acids Res 1995; 23: 1411±1418.
11. Diaz-Cano SJ, Brady SP. DNA extraction from formalin-®xed,
paraf®n-embedded tissues: protein digestion as a limiting step
for retrieval of high-quality DNA. Diagn Mol Pathol 1997; 6:
12. Diaz-Cano SJ, de Miguel M, Blanes A, Tashjian R, Galera H,
Wolfe HJ. Clonality as expression of distinctive cell kinetics
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344 Editorial
Copyright #2000 John Wiley & Sons, Ltd. J Pathol 2000; 191: 343±344.
... Core biopsies often only reflect a spatiotemporal snapshot of the whole tumor and are therefore unlikely to be fully informative about the clonal composition [123][124][125][126][127][128][129]. The size of the sample is another critical issue [130][131][132], and signal-to-noise ratios need to be balanced. One way to achieve this balance is isolate by microdissection multiple relatively small regions of tumors that more likely represent the balance of morphologically distinct units. ...
... The importance of such approach is highlighted by the observation of clustered populations within a tumor that differ in gene expression [133], as well as genetic composition [134]. However, unless large numbers of samples are provided for each tumor, this approach can easily fail to identify patches of genetically distinct cells [130][131][132]. On the other hand, larger samples, or pools of samples, lead to intermixing of small anatomically distinct units, which provides additional challenges in relation to distinguish distinct functional heterogeneity. ...
... The histopathological diagnosis should integrate molecular analysis (genome sequencing, transcriptome profiling) and protein expression profiling (especially analyses including next-generation sequencing (NGS) techniques) and be able to include gene signatures that are characteristic of a different prognosis or clinical treatment [55,70,92,94,130,[135][136][137] (Fig. 3). The incorporation of NGS and the development of new resources for the analysis of these big data, combining molecular and expression signatures, are becoming crucial for diagnosis [13,138]. ...
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In this review, we highlight the role of intratumoral heterogeneity, focusing on the clinical and biological ramifications this phenomenon poses. Intratumoral heterogeneity arises through complex genetic, epigenetic, and protein modifications that drive phenotypic selection in response to environmental pressures. Functionally, heterogeneity provides tumors with significant adaptability. This ranges from mutual beneficial cooperation between cells, which nurture features such as growth and metastasis, to the narrow escape and survival of clonal cell populations that have adapted to thrive under specific conditions such as hypoxia or chemotherapy. These dynamic intercellular interplays are guided by a Darwinian selection landscape between clonal tumor cell populations and the tumor microenvironment. Understanding the involved drivers and functional consequences of such tumor heterogeneity is challenging but also promises to provide novel insight needed to confront the problem of therapeutic resistance in tumors.
... Only informative cases (balanced allele ratios in undigested and digested controls) were included in the final analysis (10,11,13,25,26,28). Allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer; EC Apparatus Co., St Petersburg, FL), considering only allele ratios greater than 4:1 in the normalized digested lanes evidence of monoclonality. ...
... In addition, technical aspects such as PCR bias against the larger allele could contribute to preferential amplification of the smaller allele. Our DNA extraction protocol included a long protein digestion and retrieved DNA of ϳ1 kb (data not shown), excluding degraded DNA as cause (13,22,25,28,44). Our PCR design also included long denaturation and extension in the first three cycles and 7-deaza-dGTP in the amplification mixture to avoid defective amplification of CG-rich sequences (Table 1) (10,13,24,25,40). ...
... The polyclonal pattern in patients with germline RET mutation (CCH-4) needs some considerations. XCI assays can result in polyclonal patterns if the restriction endonuclease digestion is incomplete or the target DNA is hypermethylated (10,11,13,25,28). Suboptimal enzymatic digestion may change the clonality pattern of monoclonal tissues (10, 28), but that possibility was excluded keeping internal controls of endonuclease digestion. ...
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C-cell hyperplasias are normally multifocal in multiple endocrine neoplasia type 2A. We compared clonality, microsatellite pattern of tumor suppressor genes, and cellular kinetics of C-cell hyperplasia foci in each thyroid lobe. We selected 11 females from multiple endocrine neoplasia type 2A kindred treated with thyroidectomy due to hypercalcitoninemia. C-cell hyperplasia foci were microdissected for DNA extraction to analyze the methylation pattern of androgen receptor alleles and microsatellite regions (TP53, RB1, WT1, and NF1). Consecutive sections were selected for MIB-1, pRB1, p53, Mdm-2, and p21WAF1 immunostaining, DNA content analysis, and in situ end labeling. Appropriate tissue controls were run. Only two patients had medullary thyroid carcinoma foci. Nine informative C-cell hyperplasia patients showed germline point mutation in RET, eight of them with the same androgen receptor allele preferentially methylated in both lobes. C-cell hyperplasia foci showed heterogeneous DNA deletions revealed by loss of heterozygosity of TP53 (12 of 20), RB1 (6 of 14), and WT1 (4 of 20) and hypodiploid G0/G1 cells (14 of 20), low cellular turnover (MIB-1 index 4.5%, in situ end labeling index 0.03%), and significantly high nuclear area to DNA index ratio. MEN 2A (germline point mutation in RET codon 634) C-cell hyperplasias are monoclonal and genetically heterogeneous and show down-regulated apoptosis, findings consistent with an intraepithelial neoplasia. Concordant X-chromosome inactivation and interstitial gene deletions suggest clone expansions of precursors occurring at a point in embryonic development before divergence of each thyroid lobe and may represent a paradigm for other germline mutations.
... Neoplasms result from the progressive and convergent selection of cell populations for which clonality is still the hallmark indicative of acquired somatic mutations [7][8][9][10]. However, the molecular events during neoplastic transformation are not completely understood and remain essentially unknown, which leaves X-chromosome inactivation assays as the best molecular option, because this test is not based on any tumor-related genetic alteration [11,12]. ...
... Sample normalization was done in relation to the corresponding undigested sample and tissue controls. Only informative cases (2 different alleles in HhaI-undigested and HhaIdigested samples) were included in the final analysis [8,9,14,15,28]. ...
... Benign monoclonal adrenocortical lesions reveal simultaneous apoptosis downregulation and proliferation upregulation, and promote a stromal vascular reaction to support this demanding cell kinetics [14,28]. The inverted proliferation/ apoptosis relationship in monoclonal adrenocortical lesions also provides a functional basis for cellular selection, leading to clonal expansion (if proliferation predominates) or regression (if apoptosis dominates) [7,8,13,17,41,42]. The significantly increased rate of hypertetraploid cells (high 5cER correlated with monoclonal patterns) would support the presence of cycling tetraploid G 0 /G 1 cells, which suggests associated abnormalities in the anaphase checkpoint (Fig. 3). ...
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Monoclonal adrenocortical lesions show inverse correlation between proliferation and apoptosis, with proliferation being the single most important criterion of malignancy in adrenal lesions. No study yet has evaluated the variability of proliferation regarding the clonal pattern and diagnosis in adrenocortical nodular hyperplasias (ACNHs), adrenocortical adenomas (ACAs), and adrenocortical carcinomas (ACCs). We studied 69 ACNHs, 64 ACAs, and 23 ACCs (World Health Organization criteria) from 156 females. Clonality HUMARA test (from microdissected DNA samples), DNA content and proliferation analysis (slide and flow cytometry), and mitotic figure (MF) counting/50 high-power fields (HPFs) were performed in the same areas. Heterogeneity was assessed by 5cER (percentage of nonoctaploid cells with DNA content exceeding 5c) and standard deviation of MF/HPF. Statistics included analysis of variance/Student t tests regarding the clonal patterns and diagnosis. Polyclonal patterns were observed in 48 of 62 informative ACNHs and 7 of 56 informative ACAs, and monoclonal in 14 of 62 ACNHs, 49 of 56 ACAs, and 21 of 21 ACCs, with all hyperdiploid lesions (14 ACCs and 13 ACAs) being monoclonal. The standard deviation of MF/HPF progressively increased in ACNH-ACA-ACC (0.048 +/- 0.076, 0.110 +/- 0.097, 0.506 +/- 0.291, respectively; P = .0023), but did not differentiate ACNH/ACA. Only tetraploid percentage (P = .0496) and 5cER (P = .0352) distinguished polyclonal (3.64 +/- 2.20 and 0.14 +/- 0.15) from monoclonal (7.25 +/- 7.52 and 1.00 +/- 1.74) benign lesions. Malignancy significantly correlated with a low diploid percentage and high tetraploid percentage. Cell kinetic heterogeneity is the hallmark of adrenocortical neoplasms: tetraploid/hypertetraploid cell accumulation characterizes monoclonal lesions (suggesting nondisjunctional mitoses), whereas heterogeneously distributed mitotic figures and decreased diploid percentage define ACCs.
... Interpretation and inclusion criteria in each sample were achieved as follows: 7,11,21,22,26,27 (i) allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer; EC Apparatus Corp., St Petersburg, FL, USA). For evidence of loss of heterozygosity (LOH) only allele ratios ‡ 4 : 1 in any TSG were considered; otherwise retention of heterozygosity (ROH) was assigned. ...
... For evidence of loss of heterozygosity (LOH) only allele ratios ‡ 4 : 1 in any TSG were considered; otherwise retention of heterozygosity (ROH) was assigned. 7,11 This ratio represents 80% of clonal cells in the sample and was used to increase the detection specificity; 22,26,28 (ii) additional allele bands present in tumour samples but not in the corresponding controls were considered evidence of somatic single nucleotide polymorphism (SNP) by PCR ⁄ denaturing gradient gel electrophoresis. 7,19,22,29 dna sequencing ...
... Technical reasons were excluded. The sensitivity threshold of our optimized protocol was 1% for positive detection, 7,11,22,26 which applied to 100+ cell samples would result in false-negative results for DNA samples smaller than one cell equivalent. This is probably clinically irrelevant and frequently related to contamination. ...
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To correlate histological infiltration patterns with genetic and mismatch repair (MMR) profiles in muscle-invasive bladder urothelial carcinomas (UroC).Infiltration patterns were assessed in the deep compartment of muscle-invasive UroC (nodular-trabecular, 45 cases; infiltrative, 27 cases). Tumour compartment (superficial and deep to muscularis mucosa) analysis included: microsatellite pattern of TP53, RB1, WT1 and NF1 by polymerase chain reaction/denaturing gradient gel electrophoresis; mitotic, Ki67, in situ end labelling (ISEL) indices and DNA ploidy. MMR was assessed by MLH1 and MSH2 sequencing and immunohistochemistry in UroC with two or more abnormal microsatellite loci. Statistical differences were tested using anova and Fisher's exact tests. Infiltrative UroC showed lower Ki67 index 14.94 +/- 4.28, ISEL index 14.1 +/- 10.0 and shorter median survival (20 months) than nodular-trabecular UroC (Ki67 index 20.65 +/- 4.94, ISEL 20.2 +/- 22.7, 37-month survival, respectively). The genetic profile was significantly different for RB1 (P = 0.0003) and NF1 (P = 0.0023) only, being more frequently abnormal in nodular-trabecular UroC. A significant decrease in MLH1 or MSH2 protein expression with no gene mutations was identified in UroC with microsatellite abnormalities and a nodular-trabecular growth pattern.Somatic down-regulation of MMR proteins in nodular-trabecular muscle-invasive UroC results in RB1/NF1 microsatellite abnormalities, correlating with higher cellular turnover and longer survival.
... MSI can be defi ned as a change in any DNA sequence length due to either insertion or deletion of repeating units in a microsatellite within a tumor when compared to normal tissue [17,18] . The tests must be run with appropriate controls (known positive and negative controls along with the patient's normal tissue) [8,19] , which are extremely important due to the nonexceptional presence of extra-bands. The PCR approach must amplify the correct locus and accurately identify the microsatellite pattern in the patient's normal tissue. ...
... (3) PCR bias against one allele (especially the larger one in a pair) can result in preferential amplifi cation of the other allele (usually the smaller in a pair), which is the so-called artifactual allele dropout [22,23] . An appropriate extraction method, providing DNA of quality [24] , and PCR designs including both long denaturation and extension in the fi rst three cycles and 7-deaza-dGTP in the amplifi cation mixture to improve the amplification of CG-rich DNA regions, will be reasonably helpful in avoiding that bias [8,19,21,23,25] . (4) The number of polymorphic DNA regions agreed to at the NCI consensus conference includes a primary panel of at least 2 mononucleotide and 3 dinucleotide microsatellites, along with 19 alternate loci (both mono-and dinucleotides) [26] . ...
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Microsatellite instability (MSI) is a prognostic factor and a marker of deficient mismatch repair (MMR) in colorectal adenocarcinomas (CRC). However, a proper application of this marker requires understanding the following: (1) The MSI concept: The PCR approach must amplify the correct locus and accurately identify the microsatellite pattern in the patient's normal tissue. MSI is demonstrated when the length of DNA sequences in a tumor differs from that of nontumor tissue. Any anomalous expansion or reduction of tandem repeats results in extra-bands normally located in the expected size range (100 bp, above or below the expected product), differ from the germline pattern by some multiple of the repeating unit, and must show appropriate stutter. (2) MSI mechanisms: MMR gene inactivation (by either mutation or protein down-regulation as frequently present in deep CRC compartments) leads to mutation accumulation in a cell with every cellular division, resulting in malignant transformation. These mechanisms can express tumor progression and result in a decreased prevalence of aneuploid cells and loss of the physiologic cell kinetic correlations in the deep CRC compartments. MSI molecular mechanisms are not necessarily independent from chromosomal instability and may coexist in a given CRC. (3) Because of intratumoural heterogeneity, at least two samples from each CRC should be screened, preferably from the superficial (tumor cells above the muscularis propria) and deep (tumor cells infiltrating the muscularis propria) CRC compartments to cover the topographic tumor heterogeneity. (4) Pathologists play a critical role in identifying microsatellite-unstable CRC, such as occur in young patients with synchronous or metachronous tumors or with tumors showing classic histologic features. In these cases, MSI testing and/or MMR immunohistochemistry are advisable, along with gene sequencing and genetic counseling if appropriate. MSI is an excellent functional and prognostically useful marker, whereas MMR immunohistochemistry can guide gene sequencing.
... The average Lyonization ratio is close to 50:50 in large cell populations, although individual variation has been found (23), resulting in skewing toward one allele. This is the reason for using as controls for unequal Lyonization the most closely related tissue thought not to be involved in the disease process (14). ...
... No single genetic alteration of TSG proves by itself that a given proliferation is monoclonal: The LOH for that particular marker informs only on clonal expansion and cellular selection in genetically heterogeneous tumor cell populations. Only the accumulation of genetic lesions in TSG supports a monoclonal origin of tumors (15), especially if multiple samples from the same tumor show concordant genetic alteration (14,18) (see Methodological Aspects). ...
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Clonal overgrowths represent the hallmark of neoplastic proliferations, and their demonstration has been proved useful clinically for the diagnosis of malignant lymphomas based on the detection of specific and dominant immunoglobulin and/or T-cell receptor gene rearrangements. Nonrandom genetic alterations can also be used to test clonal expansions and the clonal evolution of neoplasms, especially analyzing hypervariable deoxyribonucleic acid (DNA) regions from patients heterozygous for a given marker. These tests rely basically on the demonstration of loss of heterozygosity (LOH) resulting from either hemizygosity (nonrandom interstitial DNA deletions) or homozygosity of mutant alleles observed in neoplasms. LOH analyses identify clonal expansions of a tumor cell population, and point to monoclonal proliferation when multiple and consistent LOH are demonstrated. Based on the methylation-related inactivation of one X chromosome in female subjects, X-linked markers (e.g., androgen receptor gene) will provide clonality information using LOH analyses after DNA digestion with methylation-sensitive restriction endonucleases. Therefore, both non-X-linked and X-linked analyses give complementary information, related and not related to the malignant transformation pathway respectively. Applied appropriately, these tools can establish the clonal evolution of tumor cell populations (tumor heterogeneity), identify early relapses, distinguish recurrent tumors from other metachronic neoplasms, and differentiate field transformation from metastatic tumor growths in synchronic and histologically identical neoplasms.
... Interpretation and inclusion criteria in each sample were previously reported (11,13,(21)(22)(23). Allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer, EC Apparatus Corp., St. Petersburg, FL), considering evidence of only LOH allele ratios of 4:1 or more in any TSG; otherwise, retention of heterozygosity (ROH) was assigned (13,22). ...
... Allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer, EC Apparatus Corp., St. Petersburg, FL), considering evidence of only LOH allele ratios of 4:1 or more in any TSG; otherwise, retention of heterozygosity (ROH) was assigned (13,22). This ratio represents 80% of clonal cells in the sample and was used to increase the detection specificity (10,11,23). Additional allele bands present in tumor samples, but not in the corresponding controls, were considered evidence of somatic SNP by PCR/denaturing gradient gel electrophoresis (DGGE) (13). ...
Full-text available
Despite extensive molecular investigation of adrenal pheochromocytomas, no information is available on their molecular and mismatch repair (MMR) profiles by topographic compartments. Microdissected samples from the peripheral and internal zones of 143 pheochromocytomas from a referral hospital (95 sporadic and 48 associated with multiple endocrine neoplasia type 2A) were selected for loss of heterozygosity and single nucleotide polymorphism analyses. Five polymorphic DNA regions from TP53, RB1, WT1, and NF1 were systematically studied by PCR-denaturing gradient gel electrophoresis. PATIENTS, OUTCOME MEASURES, AND INTERVENTIONS: Pheochromocytomas were classified as malignant (16 sporadic tumors with distant metastases), locally invasive (30 sporadic tumors showing retroperitoneal infiltration only), and benign (all remaining tumors). Statistical differences were evaluated using Fisher's exact test. MMR was assessed by MLH1/MSH2 sequencing and immunostaining in pheochromocytomas with two or more abnormal microsatellites. No interventions were performed in this study. Loss of heterozygosity/single nucleotide polymorphism involved TP53 in 40 of 134 informative cases (29.9%), RB1 in 22 of 106 informative cases (20.8%), WT1 in 32 of 120 informative cases (26.7%), and NF1 in 32 of 80 informative cases (40.0%). More genetic abnormalities involving the peripheral compartment were revealed in 34 pheochromocytomas (23.8%): 12 of 16 malignant, 10 of 30 locally invasive, and 12 of 97 benign. Multiple and coexistent genetic abnormalities characterized malignant pheochromocytomas (P < 0.001), whereas locally invasive pheochromocytomas showed a significantly higher incidence of NF1 alterations (P < 0.001). No mutations were identified in MLH1/MSH2, but MMR proteins significantly decreased in peripheral compartments. Multiple microsatellite alterations and topographic intratumor heterogeneity characterize malignant pheochromocytomas, suggesting a multistep tumorigenesis through somatic topographic down-regulation of MMR proteins. Locally invasive pheochromocytomas reveal topographic heterogeneity and single-locus microsatellite alterations, especially involving NF1.
... The opposite profile was observed in carcinomas, confirming the kinetic equivalency between superficial ⁄ internal and deep ⁄ peripheral compartments of neoplasms from luminal and solid organs. [5][6][7] Cell kinetics (proliferation and apoptosis) represents the basic mechanism leading to clonal expansion and tumour growth [29][30][31][32][33][34] and contributes to the theory of tumour cell topographical segregation. [5][6][7]13,14 High proliferation rates in ICs of FTA and FTHN support this theory. ...
... High-grade neoplasms show high apoptotic indices, which should be the result of the accumulation of genetic mutations reaching lethal levels for the cell. [31][32][33] Follicular tumour progression correlates with up-regulation of proliferation and relative down-regulation of apoptosis in PCs, suggesting survival and replication of genetically damaged cells leading to the accumulation of genetic mutations: 5-7,13,14 high cell proliferation would transfer mutations to daughter cells, while reduced apoptosis would allow cells carrying mutations to complete the cell cycle. ...
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The diagnosis of follicular thyroid carcinomas is mainly based on capsular and vascular invasion. The aim of this study was to determine the diagnostic relevance of nuclear features, inflammation and stromal changes.Anisokaryosis, chromatin pattern, nucleolus, nuclear pleomorphism, nuclear/cytoplasmic ratio, necrosis, stromal changes and tumour interstitial lymphocytes (TIL) were analysed in adenomatous hyperplastic nodules (39), adenomas (43) and carcinomas (28 minimally invasive, 48 widely invasive and 27 anaplastic). Ki67 immunostaining, in situ end labelling (ISEL) for apoptosis and the Ki67/ISEL index were analysed by topographical compartments. Variables were compared by histological diagnosis using Fisher's exact test, analysis of variance and Student's t-tests and considered significant if P < 0.05. TIL were absent in 96% of neoplasms and 54% of adenomatous hyperplastic nodules. Conspicuous nucleoli, increased nuclear-cytoplasmic ratio and coexistent apoptosis-myxoid changes distinguished minimally invasive carcinomas from adenomas. The most specific variables of high-grade carcinoma were vasculonecrotic patterns, nuclear hyperchromatism and pleomorphism. A kinetic advantage predominated in the internal compartments of benign lesions and in the peripheral compartments of malignant lesions.Follicular carcinomas show up-regulation of proliferation markers and the distinctive topographical kinetic profiles provide a basis for the distinction between benign and malignant and an explanation for the circumscription and encapsulation of benign lesions.
... Only informative cases (2 different alleles in control samples) were included in the final analysis and were interpreted as described. 2,12,13,30 Allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer, EC Apparatus, St Petersburg, FL). Only allele ratios of 4:1 or more in any TSG were considered evidence of loss of heterozygosity (LOH); otherwise retention of heterozygosity was assigned. ...
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We evaluated 71 muscle-invasive transitional cell carcinomas (TCCs) of the bladder by tumor compartments. Kinetic parameters included mitotic figure counting, Ki-67 index, proliferation rate (DNA slide cytometry), and apoptotic index (in situ end labeling [ISEL] of fragmented DNA using digoxigenin-labeled deoxyuridine triphosphate and Escherichia coli DNA polymerase [Klenow fragment]). At least 50 high-power fields per compartment were screened from the same tumor areas; results are expressed as percentage of positive neoplastic cells. Mean and SD were compared by tumor compartment. DNA was extracted from microdissected samples (superficial and deep) and used for microsatellite analysis of TP53 and NF1 by polymerase chain reaction-denaturing gradient gel electrophoresis. Significantly higher marker scores were revealed in the superficial compartment than in the deep compartment. An ISEL index of less than 1% was revealed in 63% (45/71) of superficial compartments and 86% (61/71) of deep compartments. Isolated NF1 alterations were observed mainly in superficial compartments, whereas isolated TP53 abnormalities were present in deep compartments. Lower proliferation and down-regulation of apoptosis define kinetically the deep compartment of muscle-invasive TCC of the bladder and correlate with the topographic heterogeneity, NF1-defective in superficial compartments and TP53-defective in deep compartments.
... The heterogeneous distribution and clustering of apoptotic cells (significantly higher ISEL-index SD) in the superficial compartments also support a sort of clonal origin for these apoptotic cells, most likely due to the accumulation of genetic abnormalities reaching cytologically lethal levels. 32,41 Likewise, the coexistence of genetic alterations in MCC supports a key role in tumorigenesis, the topographic heterogeneity resulting from the accumulation of genetic damage, partially explained by TP53 overexpression in these neoplasms. 6,37,42 This TP53 overexpression frequently correlates with mutated TP53 that partially blocks apoptosis and allows accumulation of mutations. ...
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Merkel cell carcinomas (MCC) reveal epithelial and neuroendocrine differentiation, but its topographic cell kinetics remains unknown. This study analyses proliferation, apoptosis, and DNA ploidy by topography, features that can help planning therapeutic protocols. This study topographically analyses proliferation, apoptosis, and DNA ploidy.We selected 27 small-cell MCCs (expressing one epithelial and two neural markers, with consistent ultrastructural findings) to evaluate mitotic figure counting, Ki-67 index, apoptosis index based on the in situ end labelling of fragmented DNA (using Escherichia coli DNA polymerase I, Klenow fragment), DNA ploidy, and BCL2 and TP53 immuno-expression. At least 50 high-power fields were screened per topographic compartment (superficial or papillary dermis, and deep or reticular dermis), recording average and standard deviation for each variable. Variables were statistically compared in each tumour compartment using analysis of variance and Student's t-test (significant if P < 0.05).MCCs revealed superficial aneuploid DNA content, and no topographic differences for proliferation markers. Apoptosis showed significantly lower values in the deep compartment (average, P = 0.0050, and standard deviation, P = 0.0074), correlating with increased BCL2 and TP53 immuno-expressions.High homogeneously distributed proliferation and superficial aneuploid DNA content defines MCCs. Apoptosis follows proliferation in superficial compartments, being less variable and proliferation independent in deep compartments, where it is inversely correlated with BCL2/TP53 expression.
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Although histopathologic criteria for adrenal cortical nodular hyperplasias (ACNHs) and adenomas (ACAs) have been developed, their kinetics and clonality are virtually unknown. We studied 20 ACNHs and 25 ACAs (based on World Health Organization criteria) from 45 females. Representative samples were histologically evaluated, and the methylation pattern of the androgen receptor alleles was analyzed on microdissected samples. Consecutive sections were selected for slide cytometry, flow cytometry, and in situ end labeling (ISEL). Apoptosis was studied by flow cytometry (nuclear area/DNA content plotter analysis) and by ISEL. Appropriate tissue controls were run in every case. Polyclonal gel patterns were revealed in 14/18 informative ACNHs and in 3/22 informative ACAs, whereas monoclonal gel patterns were observed in 4/18 ACNHs and 19/22 ACAs. Overlapping proliferation rates (PRs) were observed in both clonal groups, and apoptosis was detected only in G(0)/G(1) cells, especially in monoclonal ACNHs (3/4; 75%) and in polyclonal ACAs (2/3; 67%). Significantly higher PRs were observed in ACNHs with polyclonal patterns and G(0)/G(1) apoptosis and in ACAs regardless of clonality pattern and presence of G(0)/G(1) apoptosis. All except one ACNH (19/20; 95%) and 15/25 ACAs (60%) showed diploid DNA content, whereas the remaining cases were hyperdiploid. A direct correlation between PR and ISEL was observed in polyclonal lesions (PR = 29.32 ISEL - 1.93), whereas the correlation was inverse for monoclonal lesions (PR = -9.13 ISEL + 21.57). We concluded that only simultaneous down-regulated apoptosis and high proliferation result in selective kinetic advantage, dominant clone expansion, and unbalanced methylation patterns of androgen receptor alleles in ACNHs and ACAs.
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Clonality remains as the hallmark of neoplasms. A dual genetic approach using markers nonrelated (e.g., X-chromosome inactivation assays) and related to the malignant transformation (such as loss of heterozygosity analyses of tumor-suppressor genes) would provide useful clonality information from early and advanced tumor stages, respectively. Tumor progression and clonal selection would result in genetic instability and heterogeneous expression of those molecular markers related to the malignant pathway. Therefore, only the coexistence of multiple genetic abnormalities would support the clonal nature as an expression of convergent cell selection. Considering those facts, the currently available evidence on tumorigenesis and clonality in the adrenal medulla can be summarized as follows: 1. Multistep tumorigenesis defines the evolution of pheochromocytomas, as evidenced by the presence of several genetic alterations. 2. Both the significant association of nonrandom genetic alterations (specially 1p and 22q interstitial deletions) and the topographic accumulation of genetic deletions at the peripheral tumor compartment support a convergent clone selection for these neoplasms. 3. Although many genetic loci show nonrandom abnormalities, the most frequently involved locates on chromosome 1p regardless of genetic tumor background (sporadic or inherited predisposition). 4. Most pheochromocytomas should begin as monoclonal proliferations that do not always correlate with histopathologic features, particularly in inherited tumor syndromes. 5. Early histopathologic stages, described as adrenal medullary hyperplasias, are defined by hyperproliferative features in animal models and monoclonal patterns in the adrenal nodules from patients with MEN-2a.
SmtB is a member of a family of repressors which dissociate from DNA in the presence of metals; Zn2+ being the most potent inducer of metallothionein gene (smtA) transcription in vivo. In Synechococcus PCC 7942 cells devoid of chromosomal smtB, four plasmid-encoded mutants of SmtB (C61S, T11S/C14S, C121S and H105R/H106R) repressed lacZ expression driven by the smtA operator-promoter. Gel retardation assays with extracts from the complemented cells detected multiple SmtB-dependent complexes similar to those obtained with extracts from wild-type cells or with recombinant-SmtB. Elevated [Zn2+] alleviated repression in vivo by all of the mutants except H105R/H106R. These His residues (one or both) are therefore essential for Zn2+-sensing while, contrary to expectations, Cys residues are not. Hence different motifs facilitate metal-induced DNA-dissociation by SmtB and ArsR (the related oxyanion-sensing repressor), presumably generating variety in the spectra of metals sensed. Nucleotides and amino acids involved in DNA-SmtB interaction have been further defined/inferred and we also confirm that additional unknown factors form specific associations with the smt operator-promoter in elevated [Zn2+].
Several DNA extraction methods have been used for formalinfixed, paraffin-embedded tissues, with variable results being reported regarding the suitability of DNA obtained from such sources to serve as template in polymerase chain reaction (PCR)-based genetic analyses. We present a method routinely used for archival material in our laboratory that reliably yields DNA of sufficient quality for PCR studies. This method is based on extended proteinase K digestion (250 [mu]g/ml in an EDTA-free calcium-containing buffer supplemented with mussel glycogen) followed by phenol-chloroform extraction. Agarose gel electrophoresis of both digestion buffer aliquots and PCR amplification of the [beta]-globin gene tested the suitability of the retrieved DNA for PCR amplification.
Angiomyolipoma, which consists of three intimately intermixed components, smooth muscle, blood vessels, and adipose tissue, is variably considered a hamartoma, a choristoma or a true neoplasm. This study has investigated the clonality of sporadic angiomyolipomas in seven women, each with a single lesion, by determining the pattern of X-chromosome inactivation. Polymerase chain reaction (PCR) amplification of the highly polymorphic human androgen receptor gene (HUMARA) was performed on the DNA extracted from the paraffin-embedded lesional tissue microdissected to sample the admixed smooth muscle and blood vessel component (SMC/BV) and the adipose tissue component. All seven patients were heterozygous for HUMARA polymorphism upon amplification of undigested DNA from non-lesional tissue and were therefore informative for further analysis. In all patients, lesional DNA, representative of the components, was predigested with HpaII restriction enzyme for amplification of the methylated allele. In six patients, the lesions were clonal, while in one, polyclonal. The polyclonal lesion was small and had less than 20 per cent SMC/BV component. Microdissected SMC/BV component was clonal in 6/7 lesions; the scanty SMC/BV in the remaining lesion did not yield amplifiable DNA. Microdissected adipose tissue was polyclonal in all seven lesions. Angiomyolipomas are three clonal lesions due to a clonal smooth muscle cell and blood vessel component, while the polyclonal adipose tissue is probably metaplastic or reactive. Copyright © 1999 John Wiley & Sons, Ltd.
Administration of the glucocorticoid dexamethasone to adrenalectomized rats significantly decreased the serum zinc concentration within 14 hr. Dexamethasone did not detectably alter the liver zinc content, but markedly increased the proportion of zinc associated with liver metallothionein. The rate of incorporation of 35S-cystine into this protein was stimulated to a maximal extent 7 hr after administration of the glucocorticoid. Poly(A)+ mRNA from liver polysomes was isolated and translated in a cell-free protein synthesizing system. Nearly twice as much polysomal metallothionein mRNA was found 7 hr following treatment with dexamethasone. These results suggest that glucocorticoids can regulate the plasma zinc concentration by a process that is related to the biosynthesis of the hepatic zinc-binding protein, metallothionein.
Poly(A)+ (polyadenylated) mRNA coding for metallothioneins was purified 13-fold from rat liver polyribosomes and was identified by its ability to direct the biosynthesis of these proteins in a wheat-germ cell-free system. The carboxymethylated products of the protein-synthesizing system in vitro were analysed with sodium dodecyl sulphate/20% polyacrylamide-gel electrophoresis. The labelled compounds [3H]serine and [35S]cysteine were incorporated at high specific radioactivity into proteins that co-migrated with authentic metallothioneins. No [3H]leucine incorporation was found, in agreement with the amino acid composition of the metallothioneins. Metallothionein mRNA had a sedimentation coefficient of 9 S and carried a maximum of four ribosomes. At 5 h after a subcutaneous injection of ZnCl2 or CdCl2 (10 mumol/kg body wt.), the amount of this mRNA increased approx. 2- and 4-fold respectively, on the basis of translation in vitro. The increase in metallothionein mRNA (defined by translation in the wheat-germ system) was transient and, after CdCl2 treatment, fell back to control values by 17 h. Metallothioneins constituted a maximum of 0.8% of the total protein products synthesized in the wheat-germ system by total mRNA isolated from rat liver after CdCl2 treatment.
In both large and small intestine, mutagen administration leads to the occurrence of isolated crypts that are completely populated by a mutated phenotype; therefore, it has been proposed that crypts are maintained by a single stem cell. We show in mice that a single dose of mutagen leads to an early transient increase in frequency of colonic crypts that show a partial mutated phenotype and a later increase in frequency of crypts that show a complete mutated phenotype. This increase reaches a plateau at about the same time as the disappearance of partially mutated crypts. The same is true in the small intestine, but the time course is much slower. We propose an explanation based on multiple crypt stem cells that occupy a "stem cell niche," with random cell loss after stem cell division. A small difference in the number of crypt stem cells that occupy the niche provides a simple explanation for the surprisingly large difference in the time course of phenotypic changes in the large and small intestines after administration of a single dose of mutagen.